Control of the worldwide HIV epidemic will require development of a preventive and therapeutic HIV vaccine. An effective vaccine will need to elicit neutralizing antibodies as well as an effective CTL response. Toll-like receptor (TLR) ligands are potent activators of antigen presenting cells that are central in the development of a successful adaptive immune response. We hypothesize that using synthetic TLR ligands, such as immunostimulatory DNA, in HIV vaccine preparations will improve anti-HIV immune responses. Furthermore, inhibiting TLR-induced inhibitory pathways that down-regulate immune responses, such as IL-10 and indoleamine 2,3-dioxygenase (IDO), should further optimize immune responses. Thus, we propose to investigate: 1) the development of memory CD8+ T cell responses after TLR ligand-based vaccination with a model antigen (ovalbumin) and with inactivated SIV, characterization of the effector and central memory responses, and requirement for CD4+ T cells in the development and maintenance of memory cells; 2) the ability to augment immune responses by inhibiting IDO, a critical enzyme in tryptophan catabolism that down-regulates T cell responses, and/or by neutralizing the inhibitory cytokine IL-10 in in vitro and in vivo systems; 3) the outcome of the approaches mentioned above to elicit immune responses against SIV in mice that will be reconstructed to express the human/primate cellular expression profile of TLR9; 4) the most promising approaches selected in SA 1-3 in an in vitro and in an in vivo macaque system before and after SIV challenge; 5) the efficacy of TLR ligand-based vaccines in a human PBMC system. Specifically, the augmentation of CD4+ and CD8+ T cell memory responses to CMV in CMV+ donors after incubation with TLR ligands and inactivated CMV will be assessed as well as the effect of CD4-depletion on the anti-CMV responses and anti-HIV responses by using PBMCs from HIV+/CMV+ donors. These experiments were designed to provide a balanced investigation that explores fundamental immunology and provides the practical aspects of vaccinology that will move us closer to development of an effective HIV vaccine for humans.